A “species” should be understood to be a chemical species such as a molecule or a complex, or a physical object such as a nanoparticle. “Reversibly photoswitchable species” should be understood to mean a species exhibiting at least two distinct states, having different fluorescence properties, and being able to switch from one state to the other reversibly by the effect of light. Examples of reversibly photoswitchable fluorescent species are the protein “Dronpa” and the complex “Spinach—DFHBI” (“Spinach” being an aptamer of RNA and DFHBI being a fluorogenic probe). These species can, in particular, be used as probes or markers.
Imaging, and more particularly fluorescence microscopy, has become an essential tool in biology, but also in other disciplines such as materials science. Its applications, however, are limited by the capacity to observe a signal of interest in a background of fluorescence or of noise. This problem is particularly acute in in vivo imaging applications, in which the fluorescent markers to be detected are scattered in a complex self-fluorescent and/or light-scattering medium; the useful signal is then embedded in an intense background noise.
Another limitation of the conventional fluorescence detection and imaging techniques lies in the fact that numerous fluorophores exhibit wide emission bands; consequently, it is difficult to selectively detect a number of fluorescent markers in one and the same sample, because their emission spectra tend to be superimposed.
To overcome these limitations, it has been proposed to use reversibly photoswitchable fluorescent probes, an illumination that is modulated (variable in time in a predetermined manner) and a demodulation of the detected fluorescence signal. That makes it possible to exploit the temporal dynamics of a reversibly photoswitchable probe—which is specific to it and is different from that of interfering fluorophores—to extract a useful signal from the background noise; this is then called “dynamic contrast”.
One technique known from the prior art exploiting this principle is known by the name of OLID, an acronym for “Optical Lock-In Detection”. It is described in the article by G. Marriott et al. “Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells”, PNAS, vol. 105, no. 46, pages 17789-17794 (18 Nov. 2008). One drawback with this technique is that it does not provide quantitative information on the concentration of the reversibly photoswitchable fluorophore. Also, it requires a light excitation sequence with two colors and at least one reference pixel.
Another technique known from the prior art using a photoswitchable fluorescent probe and a modulated excitation is known by the name of SAFIRe, an acronym for “Synchronously Amplified Fluorescence Image Recovery”. It is described in the article by Ch. I. Richards et al. “Synchronously Amplified Fluorescence Image Recovery (SAFIRe)”, J. Phys. Chem. B 2010, 114, 660-665. This technique also uses a two-color excitation. The optimization of the dynamic contrast has the drawback of being done empirically, which introduces an additional implementation complexity.
The article by Q. Wei and A. Wei “Optical Imaging with Dynamic Contrast Agents”, Chem. Eur. J., 17, 1080-1091 reports on a number of known dynamic contrast techniques. In addition to the abovementioned OLID and SAFIRe techniques, based on an optical modulation of the fluorescence, this article describes techniques that exploit a magnetomotive or photothermal modulation. These techniques are complex to implement, precisely because they require both an optical system for the excitation and the detection of the fluorescence and a non-optical (magnetic or thermal) modulation system.
Thus, all the fluorescence detection techniques known from the prior art exploiting a dynamic contrast have the drawback of a relatively complex implementation. Furthermore, none of them offers sufficient selectivity to allow for the successive detection of a significant number (of the order of 10, even more) of fluorescent species in one and the same sample. Moreover, these techniques have been developed exclusively for microscopic applications and cannot be easily transposed to the remote sensing of fluorescent species in the environment.